In the bleeding individual, coagulation is initiated by the Tissue Factor (TF)/activated Factor VII (FVIIa) complex when extravascular TF is exposed to FVIIa in the blood. TF/FVIIa complex formation leads to the activation of Factor X (FX) to FXa which, together with activated Factor V (FVa), generates a limited amount of thrombin. The initial amount of thrombin activates platelets which, in turn, result in the surface exposure of platelet phospholipids that support the assembly and binding of the tenase complex, consisting of activated Factor VIII (FVIIIa) and activated Factor IX (FIXa). The tenase complex is a very efficient catalyst of FX activation and FXa generated in this step serves as the active protease in the FVa/FXa pro-thrombinase complex which is responsible for the final thrombin burst. Thrombin cleaves fibrinogen to generate fibrin monomers, which polymerise to form a fibrin network. The rapid and extensive thrombin burst is a prerequisite for the formation of a solid and stable fibrin clot.
An inadequate propagation of FXa and thrombin generation caused by FVIII or FIX deficiency is the underlying reason for the bleeding diathesis in haemophilia A or B patients, respectively. In people with haemophilia, FXa generation is primarily driven by the TF/FVIIa complex: FVIII or FIX deficiency leads to rudimentary FXa generation by the tenase complex. TF/FVIIa-mediated activation of FX to FXa is, however, temporary because tissue factor pathway inhibitor (TFPI) inhibits the TF/FVIIa complex in an auto-regulatory loop. Feed-back inhibition leads to formation of the TF/FVIIa/FXa/TFPI complex. Blunting of TFPI inhibition prolongs TF/FVIIa-mediated activation of FX during initiation of coagulation and promotes haemostasis. Haemophilia patients suffer from an impaired tenase activity due to, e.g., FVIII or FIX deficiency. Blocking of TFPI inhibition can in these patients compensate for an inadequate FXa generation and normalize the bleeding diathesis.
TFPI is a slow, tight-binding competitive inhibitor which regulates FX activation through inhibition of both FXa and the TF/FVIIa complex. TFPI contains three tandemly arranged Kunitz-type Protease Inhibitor domains (KPI 1-3). TFPI inhibition of FXa occurs in a biphasic reaction which initially leads to a loose TFPI-FXa complex, which then slowly rearranges into a tight binding TFPI-FXa complex, where KPI-2 binds and blocks the active site of FXa. Following initiation of coagulation, TF/FVIIa-mediated FXa generation is tightly down-regulated by TFPI. TF/FVIIa is inhibited by TFPI in a process which, as a rate limiting step, appears to involve TFPI inhibition of FXa, either when FXa is bound to the TF/FVIIa complex or bound in the near vicinity of the TF/FVIIa complex on the cell membrane (Baugh et al., J Biol Chem. 1998; 273(8):4378-86). KPI-1 contributes to the formation of the tight TFPI-FXa complex and directly binds and also blocks the active site of TF-bound FVIIa (Girard et al., Nature 1989; 338:518-520; Peraramelli et al., Thromb. Haemost. 2012; 108:266-276).
In vivo, TFPI is found in several cellular compartments. A major fraction of TFPI is associated with the vascular endothelium and a minor fraction circulates in the blood. Two splice variants of TFPI, TFPI alpha (TFPIα) and TFPI beta (TFPIβ), have been described to be present in humans. Endothelial cells are the major sites for TFPI production and express both variants. The predominant form on the endothelial cell surface is presumably TFPIβ. TFPIα is either secreted into the plasma or found in intracellular stores, which can be released upon certain stimuli. Secreted TFPIα circulates in the blood either as a full-length protein (10%) or as modified proteins with different molecular masses (90%); the latter may be due to, e.g., truncation of the C-terminal region or association with lipoproteins. Secreted TFPIα may also bind to the endothelial cells surface through interactions with e.g. glycoaminoglycans. TFPIα is also produced by and stored in platelets.
The half-life of TFPI in humans is known to be about 60-120 minutes and the total normal human plasma TFPI concentration is known to be about 1.0-2.5 nM. In contrast, the half-life of antibodies in humans is known to be long: up to several weeks, depending on the immunoglobulin subtype, origin and specific amino acid composition. The total TFPI concentration (free TFPI plus TFPI/antibody complexes) in plasma may rise to concentrations as high as 20-40 nM (Augustsson et al., J Thromb Haemost. 2013, 11:s2, PA 4.14-2) when certain known TFPI antibodies, such as the mAb 0001 antibody disclosed in Augustsson et al. supra, forms a complex with TFPI in the circulation. In vivo administration of TFPI antibodies may, therefore, cause TFPI/antibody complexes to accumulate to concentrations that are much higher than the pre-dose plasma concentration of TFPI. Accumulation may, in cases where the inhibitory activity of TFPI/antibody complexes is not fully neutralised, reverse the effect of an otherwise procoagulant TFPI antibody resulting in a net anticoagulant effect.
The inventors envisage that the antibodies disclosed herein, and pharmaceutical compositions comprising them, may address such limitations.